Bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, SEQ ID NO:1) is a vasoactive peptide released from the precursor plasma kininogens by plasma and tissue kallikreins and other enzymes (Silva et al., Amer. J. Physiol. 156: 261-274 (1949)). The parent proteins of bradykinin, high (HK) and low (LK) molecular weight kininogens are recognized to have the ability to inhibit α- and γ-thrombin-induced platelet activation (Meloni et al., J. Biol. Chem. 266, 6786 (1991); Puri et al., Blood 77, 500 (1991)). Both low and high molecular weight kininogens have identical amino acid sequences from their amino-terminus through 12 amino acids beyond the carboxy-terminus of bradykinin. Both LK and HK share a common heavy chain (62 kDa), the bradykinin (BK) moiety (0.9 kDa), and the first 12 amino acids of the amino terminal portion of each of their “light chains” (Takagaki et al., J. Biol. Chem. 260, 8601-8609 (1985); Kitamura et al., J. Biol. Chem., 260, 8610-8617 (1985)). This identity corresponds to residues 1 through about residue 383 (See Salveson et al., Biochem J. 243, 429 (1986); Kellerman et al., Eur. J. Biochem. 154, 471 (1986)). The HK and LK kininogens diverge in the size of their light chains; the light chain of LK is 4 kDa; that of HK is 56 kDa. (Takagaki et al., supra; Kitamura et al., supra.). The kininogens prevent thrombin-induced platelet activation. Full-length kininogens prevent thrombin from binding to platelets. Thus, the prior art indicated that kininogens' ability to inhibit thrombin activation of platelets was more than direct interaction with the thrombin molecule itself (Meloni et al., supra; Puri et al., supra).
The thrombin inhibitory activity of the kininogens was thought to be localized to an isolated domain 3 of the kininogens' heavy chain, because domain 3 retained all the thrombin inhibitory activity of the whole protein (Jiang et al., J. Biol. Chem. 267, 3712 (1992)). The thrombin inhibitory activity of the kininogens was later found to be associated with domain 4, the bradykinin sequence, which was attached to the carboxyterminal end of isolated domain 3 prepared by proteolytic cleavage of whole LK (Hasan et al., Circulation 94, 517-528 (1996); Tayeh et al., J. Biol. Chem. 269, 16318-16325 (1994)). Bradykinin, itself, has been recognized to antagonize the effects of α-thrombin (Ehringer et al., Inflammation. 21:279-298 (1997)). The thrombin inhibitory region of domain 4, the bradykinin sequence, demonstrated a number of features. This sequence did not prevent thrombin from binding to platelets and it did not prevent the thrombin receptor activation peptide (TRAP), SFLLRN (Ser-Phe-Leu-Leu-Arg-Asn, SEQ ID NO:2), from stimulating calcium mobilization and platelet aggregation in platelets. This sequence from domain 4 prevented thrombin-activated platelets from losing an epitope to monoclonal antibody SPAN12. Monoclonal antibody SPAN12 is directed to the thrombin cleavage site on protease activated receptor 1 (PAR1) (Hasan et al., supra; Vu et al., Cell 64, 1057-1068 (1991); Brass et al., J. Biol. Chem. 267, 13795-13798 (1992)). Monoclonal antibody SPAN12 was raised to the peptide NATLDPRSFLLR (Asn-Ala-Thr-Leu-Asp-Pro-Arg-Ser-Phe-Leu-Leu-Arg, SEQ ID NO:3) (Brass et. al., supra.). Further, bradykinin analog peptides prevented α-thrombin from cleaving the peptide NATLDPRSFLLR (SEQ ID NO:3) between arginine and serine, the identical place on PAR1 that thrombin cleaves to activate this receptor. Although there are a number of peptide analogs of bradykinin that demonstrate thrombin inhibiting activity against platelet activation, the minimal sequences retaining this activity are the peptides, RPPGF (Arg-Pro-Pro-Gly-Phe, SEQ ID NO:4), RPPG (Arg-Pro-Pro-Gly, SEQ ID NO:5), and RPP (Arg-Pro-Pro). FITC-labeled (fluorescein isothiocyanate) RPPGF (SEQ ID NO: 4) has the ability to directly bind to platelets (Hasan et al., Thromb Haemost. 82, 1182-1187 (1999)). These data indicate that the RPPGF (SEQ ID NO:4) and related bradykinin analog peptides have the ability to bind to platelets to prevent thrombin-induced platelet activation. RPPGF (SEQ ID NO:4) and its related peptide, MAP4-RPPGF (β-Ala-Lys-2Lys-4(Arg-Pro-Pro-Gly-Phe)) (RPPGF disclosed as SEQ ID NO: 4) have the ability to interfere with α- or γ-thrombin-induced platelet activation two ways: at high concentrations these peptides are retrobinders to the active site of thrombin (Ki=1.75 mM). At lower concentrations they bind to protease activated receptor 1 (PAR1) near the thrombin cleavage site to prevent thrombin cleavage of the extracellular domain of PAR1 (Hasan et al., Blood. 98, 530a, (2001); Hasan et al. Amer J Physiol. Heart Circ Physiol. 285, H183, (2003)). Bradykinin, itself, has been shown to be a direct inhibitor of thrombin with a Ki between 170 to 326 μM (Cleary et al., Arch. Biochem. Biophys. 410, 96-106 (2003)). Furthermore, rOicPGF and MAP4-rOicPGF (β-Ala-Lys-2Lys-4(rOicPGF)) inhibit the enzymatic activity of α-thrombin and factor VIIa (Nieman et al. J. Pharm Exp Therap. 311, 492 (2004)). Thrombin has two binding sites on PAR1. It binds by its exosite I region to a hirugen-like region on the carboxyterminus of the extracellular fragment of PAR1 which includes the amino acid sequence Asp-Lys-Tyr-Glu-Pro-Phe-Trp-Glu-Asp-Glu-Glu-Lys (SEQ ID NO:6) (Ayala et al. Proteins:Structure, Function, and Genetics. 45, 107-116 (2001)). It also binds to a region adjacent to the thrombin cleavage site on PAR1, the sequence Leu-Asp-Pro-Arg (SEQ ID NO:7) (Ayala et al. Proteins:Structure, Function, and Genetics. 45, 107-116 (2001)). Alternatively, when thrombin cleaves PAR4, it only binds to a region adjacent to the thrombin cleavage site Leu-Pro-Ala-Pro-Arg (SEQ ID NO:8) (Ayala et al. Proteins:Structure, Function, and Genetics. 45, 107-116 (2001)). On human PAR4, there is no equivalent hirugen binding region as seen on PAR1 on the extracellular fragment of PAR4. Both RPPGF (SEQ ID NO:4) and rOicPGF prevent RPPGFK-biotin (SEQ ID NO: 15) from binding to a peptide (Ser-Ile-Leu-Pro-Ala-Pro-Arg-Gly-Tyr-Pro-Gly-Gln, (SEQ ID NO:9)) of the thrombin cleavage site on the exodomain of human protease activated receptor 4 (PAR4) (Nieman et al. FEBS Letters, 579, 25, (2005)). RPPGF (SEQ ID NO:4) binds via its arginine to the exodomain of human PAR4 to prevent thrombin cleavage (Nieman et al. FEBS Letters, 579, 25, (2005)). If proline 46 on the exodomain of PAR4 is changed to an alanine, RPPGF (SEQ ID NO:4) does not bind to the recombinant protein (Nieman et al. FEBS Letters, 579, 25, (2005)). This information indicates that the arginine on RPPGF (SEQ ID NO:4) specifically binds to Pro46 on the exodomain of human PAR4 to prevent α-thrombin cleavage (Nieman et al. FEBS Letters, 579, 25, (2005)).
The importance of the use of RPPGF (SEQ ID NO:4) and related compounds has been shown in animal studies. RPPGF (SEQ ID NO:4) prevents coronary thrombosis in the canine electrolytic injury model similar to aspirin treatment (Hasan et al. Thrombosis and Haemostasis 82, 1182-1187 (1999)). MAP4-RPPGF (β-Ala-Lys-2Lys-4(Arg-Pro-Pro-Gly-Phe)) (RPPGF disclosed as SEQ ID NO: 4) prevents cyclic flow variations in the Folt's model for canine coronary thrombosis to a similar degree as aspirin or clopidogrel (Hasan et al. Thrombosis and Haemostasis 86, 1296-1304 (2001)). RPPGF (SEQ ID NO:4) infusion delays the time to death in lipopolysaccharide-treated rats (Morinelli et al. J. Pharm Exp. Ther. 296, 71-76 (2001)). RPPGF (SEQ ID NO:4) reduced platelet activation and deposition in an ex vivo model of balloon injury to the vessel wall similar to the effects of aspirin (Prieto et al. Cardiovascular Research. 53, 984-992 (2001)). Finally, MAP4-RPPGF (RPPGF disclosed as SEQ ID NO: 4) delays the time to thrombosis of the mouse carotid artery and inhibits mouse platelet aggregation (Srikanth et al. Blood. 100, 24a, (2002), Nieman et al. J. Pharm Exp Therap. 311, 492 (2004)).
More physiologic investigations have been performed on RPPGF (SEQ ID NO:4). RPPGF (SEQ ID NO:4) has been recognized as the terminal breakdown product of angiotensin converting enzyme (Kuoppala et al. Am J Physiol Heart Circ Physiol. 278, H1069 (2000), Murphey et al. J Pharm Exp Therap. 294, 263 (2000), Murphey et al. Anal Biochem. 292, 87 (2001)). Infusion of RPPGF (SEQ ID NO:4) into rats ameliorated the deleterious effects of lipopolysaccharide (Morinelli et al. J Pharm Exp Therap. 296, 71 (2001)). Furthermore, treatment of rats with RPPGF (SEQ ID NO:4) resulted in reduction of local thrombin-induced edema in their brains (Jiang et al. J. Cerebral Blood Flow & Metabolism. 22, 404 (2002)). RPPGF (SEQ ID NO:4) and its analog rOicPGF block biotin-RPPGF (SEQ ID NO:4) from binding to a recombinant exodomain of human protease activated receptor 1 (Nieman et al. J Pharm Exp Therap. 311, 492 (2004)). D-Arginine-Oic-Pro-Gly-Phe inhibits thrombin-induced mouse platelet aggregation, prolongs mouse bleeding times, and inhibits mouse carotid artery thrombosis (Nieman et al. J. Pharm Exp Therap. 311, 492 (2004)).
The present invention relates to inhibition of thrombin-induced activation in human cells. Inhibition of thrombin activation of platelets or other cells can be either through an inhibitor of thrombin directed to the thrombin molecule itself or an inhibitor directed to substrates of thrombin. PAR1 and PAR4 (Xu et al. Proc. Natl. Acad. Sci. 95, 6642, (1998) are specific substrates of thrombin to which this class of inhibitors are directed. The present invention is directed to inhibition of activation of these thrombin substrates on any cell that expresses PAR1 or PAR4. These cells include normal platelets, endothelial cells, smooth muscle cells, fibroblasts, neuronal cells, or any other normal or cancerous cell that contains these receptors. U.S. patent application Ser. No. 10/426,968, filed May 1, 2003 describes selective peptide inhibitors of thrombin and activation of PAR1 and PAR4.
The following abbreviations have been used:                A: any naturally occurring amino acid or a synthetic amino acid as shown in Table I        BK: bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, SEQ ID NO:1);        D3: domain 3 of kininogens;        D4: domain 4 of kininogen that is the bradykinin region;        FITC: fluorescein isothiocyanate;        HK: high molecular weight kininogen;        LK: low molecular weight kininogen;        MAP4-RPPGF: A four-branched peptide consisting of a β-alanine core with a single lysine attached at its amino terminal end followed by two additional lysines. Each lysine will then have two RPPGF (SEQ ID NO:4) peptides attached by the phenylalanine to each of the lysines;        NAT12: peptide sequence Asn-Ala-Thr-Leu-Asp-Pro-Arg-Ser-Phe-Leu-Leu-Arg (SEQ ID NO:3) that spans the α-thrombin cleavage site on the thrombin receptor;        SIL12 peptide sequence Ser-Ile-Leu-Pro-Ala-Pro-Arg-Gly-Tyr-Pro-Gly-Gln (SEQ ID NO:9) that spans the α- and γ-thrombin cleavage site on the thrombin receptor.        PAR1: protease activated receptor 1;        PAR4 protease activated receptor 4;        PTCA: percutaneous transluminal coronary angioplasty;        RPPGF: Arg-Pro-Pro-Gly-Phe (SEQ ID NO:4);        RPPGC: Arg-Pro-Pro-Gly-Cys (SEQ ID NO:10)        rOicPGF: D-Arg-Oic-Pro-Gly-Phe;        MAP4-rOicPGF: β-Ala-Lys-2Lys-4(D-Arg-Oic-Pro-Gly-Phe);        FPRPG: Phe-Pro-Arg-Pro-Gly (SEQ ID NO:11)        SPAN12: a monoclonal antibody specific for the sequence Asn-Ala-Thr-Leu-Asp-Pro-Arg-Ser-Phe-Leu-Leu-Arg (SEQ ID NO:3) that spans the α-thrombin cleavage site on PAR1; and        X: nomenclature for one of eight synthetic amino acids        Z: nomenclature for any naturally occurring amino acid.        APTT activated partial thromboplastin time, an assay to measure the clotting of plasma.        PT prothrombin time, an assay to measure the clotting of plasma        TCT thrombin clotting time, an assay to measure the integrity of fibrinogen in plasma or with purified fibrinogen        